PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens, a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

Tobacco introduced Perilla frutescens and Ocimum basilicum genes attenuates neutrophilic inflammation in lung tissues of COPD rats.

The new-type tobacco varieties "Zisu" and "Luole" were obtained by distant hybridization between N. tabacum L. var. HHY and Perilla frutescens and Ocimum basilicum, with obviously different chemical composition. Smoking is the major risk factor for COPD, characterized by neutrophil-dominant inflammation. In the present study, rat COPD model was established by cigarette exposure, and the health hazard of three varieties was compared by general condition observation, pathological and morphological evaluation, total and differential cell numeration, and characterization of major inflammatory mediators and MAPK/NF-κB pathway, etc. Rats in "HHY" group developed obvious symptoms such as cough, dyspnea, mental fatigue, etc., but these symptoms were obviously mitigated in "Zisu" and "Luole" groups. H&E staining analysis, including score, MLI, MAN, wt% and WA%, showed that "Zisu" and "Luole" significantly alleviated lung injury and the degree of airway remodeling and emphysema compared to "HHY". In BALF, the number of total leukocyte and the percent neutrophils in "Zisu" and "Luole" groups were evidently lower than "HHY" group. The levels of inflammatory mediators, such as IL-8, MPO, MIP-2, LTB4, TNF-α and neutrophil elastase, in "HHY" group were obviously higher than "Zisu" and "Luole" groups. The ROS-mediated NF-κB p65 and p38MAPK pathways may play an important role. Results indicated that tobacco introduced perilla and basil genes could remarkably attenuate recruitment, infiltration and activation of neutrophils and intervene in airway inflammation, retarding disease progression, especially "Zisu". Changes in chemical composition via breeding techniques may be a novel way for tobacco harm reduction.

Genome-Wide Analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Family in Perilla frutescens and Functional Characterization of PfGPAT9 Crucial for Biosynthesis of Storage Oils Rich in High-Value Lipids.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol (TAG) biosynthesis. However, GPAT members and their functions remain poorly understood in Perilla frutescens, a special edible-medicinal plant with its seed oil rich in polyunsaturated fatty acids (mostly α-linolenic acid, ALA). Here, 14 PfGPATs were identified from the P. frutescens genome and classified into three distinct groups according to their phylogenetic relationships. These 14 PfGPAT genes were distributed unevenly across 11 chromosomes. PfGPAT members within the same subfamily had highly conserved gene structures and four signature functional domains, despite considerable variations detected in these conserved motifs between groups. RNA-seq and RT-qPCR combined with dynamic analysis of oil and FA profiles during seed development indicated that PfGPAT9 may play a crucial role in the biosynthesis and accumulation of seed oil and PUFAs. Ex vivo enzymatic assay using the yeast expression system evidenced that PfGPAT9 had a strong GPAT enzyme activity crucial for TAG assembly and also a high substrate preference for oleic acid (OA, C18:1) and ALA (C18:3). Heterogeneous expression of PfGPAT9 significantly increased total oil and UFA (mostly C18:1 and C18:3) levels in both the seeds and leaves of the transgenic tobacco plants. Moreover, these transgenic tobacco lines exhibited no significant negative effect on other agronomic traits, including plant growth and seed germination rate, as well as other morphological and developmental properties. Collectively, our findings provide important insights into understanding PfGPAT functions, demonstrating that PfGPAT9 is the desirable target in metabolic engineering for increasing storage oil enriched with valuable FA profiles in oilseed crops.

Genome-wide comprehensive characterization and transcriptomic analysis of AP2/ERF gene family revealed its role in seed oil and ALA formation in perilla (Perilla frutescens).

Perilla (Perilla frutescens) is a potential specific oilseed crop with an extremely high α-linolenic acid (ALA) content in its seeds. AP2/ERF transcription factors (TFs) play important roles in multiple biological processes. However, limited information is known about the regulatory mechanism of the AP2/ERF family in perilla's oil accumulation. In this research, we identified 212 AP2/ERF family members in the genome of perilla, and their domain characteristics, collinearity, and sub-genome differentiation were comprehensively analyzed. Transcriptome sequencing revealed that genes encoding key enzymes involved in oil biosynthesis (e.g., ACCs, KASII, GPAT, PDAT and LPAAT) were up-regulated in the high-oil variety. Moreover, the endoplasmic reticulum-localized FAD2 and FAD3 were significantly up-regulated in the high-ALA variety. To investigate the roles of AP2/ERFs in lipid biosynthesis, we conducted a correlation analysis between non-redundant AP2/ERFs and key lipid metabolism genes using WGCNA. A significant correlation was found between 36 AP2/ERFs and 90 lipid metabolism genes. Among them, 12 AP2/ERFs were identified as hub genes and showed significant correlation with lipid synthase genes (e.g., FADs, GPAT and ACSL) and key regulatory TFs (e.g., LEC2, IAA, MYB, UPL3). Furthermore, gene expression analysis identified three AP2/ERFs (WRI, ABI4, and RAVI) potentially playing an important role in the regulation of oil accumulation in perilla. Our study suggests that PfAP2/ERFs are important regulatory TFs in the lipid biosynthesis pathway, providing a foundation for the molecular understanding of oil accumulation in perilla and other oilseed crops.

Enhancing the potential for cadmium phytoremediation by introducing Perilla frutescens genes in tobacco.

To improve the potential of cadmium phytoremediation, distant hybridization between tobacco (Nicotiana tabacum L. var. 78-04), a high-biomass crop, and Perilla frutescens var. frutescens, a wild Cd-hyperaccumulator, was carried out, developing a new variety N. tabacum L. var. ZSY. Seedlings at the six-leaf stage were grown in hydroponics and treated with 0 (control), 10 µM, 180 µM, and 360 µM CdCl2 for 7 days; then, the differences in Cd tolerance and accumulation and physiological and metabolic responses were evaluated among "ZSY" and its parents. At high Cd dose, the growth of "ZSY," such as fresh weight, plant height, and root length, was evidently better than "78-04." In contrast to P. frutescens and "78-04," "ZSY" could accumulate more Cd in shoots than roots. Under the same treatment, "ZSY" accumulated greater amounts of Cd in both shoots (195-1523 mg kg-1) and roots (140-1281 mg kg-1) than "78-04" (shoots: 35-89 mg kg-1, roots: 39-252 mg kg-1), followed by P. frutescens (shoots: 156-454 mg kg-1, roots: 103-761 mg kg-1). BCF and TF values of "ZSY" reached 38-195 and 1.2-1.4, which were far higher than those of "78-04" (BCF: 2.2-35.3, TF: 0.35-0.9). Perilla frutescens was found with BCF and TF of 11-156 and 0.5-1.5. Cd stress obviously promoted the production of ROS and MDA in seedlings but reduced chlorophyll contents, especially in "78-04." As a response to Cd stress, "ZSY" had higher SOD and CAT activities when compared to P. frutescens and "78-04," while "78-04" produced more POD and proline than those of P. frutescens and "ZSY." Cd stress could affect the production and accumulation of alkaloids and phenolic compounds in root (endodermis and cortex) and mesophyll. At high Cd doses, P. frutescens and "ZSY" had more alkaloids in tissues than "78-04." Phenolic compounds in "78-04" were more obviously inhibited compared with P. frutescens and "ZSY." These secondary metabolites may play an important role in eliminating oxidative damage and enhancing Cd tolerance and accumulation in "ZSY" and P. frutescens. Results indicated that distant hybridization could be one of effective methods for introducing excellent genes from metal-hyperaccumulators into high biomass species, creating plants with superior phytoremediation potential.

An Ethanol Extract of Perilla frutescens Leaves Suppresses Adrenergic Agonist-Induced Metastatic Ability of Cancer Cells by Inhibiting Src-Mediated EMT.

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

[Cloning and functional characterization of a lysophosphatidic acid acyltransferase gene from Perilla frutescens].

Perilla (Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2. The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E. coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E. coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco (Nicotiana tabacum, variety: Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E. coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.

A Methoxyflavanone from Perilla frutescens Induces Cellular Senescence in A549 Human Lung Adenocarcinoma Cells but Not in Normal Human Bronchial Epithelial Cells.

Cellular senescence is an inherent tumor suppressive process, and cancer-targeted senescence induction represents an attractive anti-tumor strategy. Here, we show that a methoxyflavanone derivative (Perilla-derived methoxyflavanone, PDMF) from the Asian medicinal herb, Perilla frutescens, induces cellular senescence in A549 human adenocarcinoma cells but not in normal human bronchial epithelial (NHBE) cells. We also provide evidence that PDMF preferentially activates the p53-p21 pathway in A549 cells, and that p53 is essential for its pro-senescent activity.

Perilla frutescens seed oil combined with Anredera cordifolia leaf powder attenuates age-related cognitive decline by reducing serum triglyceride and glucose levels in healthy elderly Japanese individuals: a possible supplement for brain health.

We have shown that Anredera cordifolia extract improves learning and memory in a senescence-accelerated mouse model, and that α-linolenic acid (ALA)-rich Perilla frutescens seed oil (PO) improves brain function in healthy Japanese adults and elderly individuals. Herein, we present a 12-month, randomised, double-blind, parallel-armed intervention trial examining the effects of PO supplementation alone or in combination with A. cordifolia leaf powder on brain function in healthy elderly Japanese individuals. Participants were randomly divided into two groups: the PO group received 1.47 mL PO (0.88 g ALA) daily via soft gelatine capsules, and the POAC group received 1.47 mL PO and 1.12 g A. cordifolia leaf powder (1.46 mg vitexin and 1.12 mg adenosine) daily. After 12 months of intervention, the POAC group showed generally higher cognitive index scores than the PO group. The beneficial effects of combined supplementation on cognitive function were associated with increased ALA and eicosapentaenoic acid levels in red blood cell plasma membranes, increased serum biological antioxidant potential, and decreased serum triglyceride, glucose, and N-(epsilon)-carboxymethyl-lysine (CML), an advanced glycation end-product and biochemical marker of oxidative stress levels. The effects of combined supplementation on cognitive function also showed a significant negative correlation with serum CML levels after 12 months of intervention. Our findings suggest that combined long-term supplementation with PO and A. cordifolia more effectively ameliorates age-related cognitive decline than PO alone. These findings may serve as a basis for the development of new supplements for brain health. Clinical Trial Registry, UMIN000040863.

The Essential Oil Derived from Perilla frutescens (L.) Britt. Attenuates Imiquimod-Induced Psoriasis-like Skin Lesions in BALB/c Mice.

Psoriasis is reported to be a common chronic immune-mediated skin disease characterized by abnormal keratinocytes and cell proliferation. Perilla leaves are rich in essential oils, fatty acids, and flavonoids, which are recognized for their antioxidant and anti-inflammatory effects. In this study, the alleviating effect of essential oil (PO) extracted from Perilla frutescens stems and leaves on imiquimod (IMQ) -induced psoriasis-like lesions in BALB/c mice were investigated. Results showed that PO ameliorated psoriasis-like lesions in vivo, reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly-6G), which is a marker of neutrophil activation, and inhibited the expression of inflammatory factors interleukin 1 (IL-1), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2). In addition, PO significantly decreased the expression of cytokines such as IL-6, IL-1, interleukin 23 (IL-23), interleukin 17 (IL-17), and nuclear factor kappa-B (NF-κB). Furthermore, the down-regulation of mRNA levels of psoriasis-related pro-inflammatory cytokines, such as IL-17, interleukin 22 (IL-22), IL-23, interferon-α (IFN-α), and Interferon-γ (IFN-γ) was observed with the treatment of PO. All results show a concentration dependence of PO, with low concentrations showing the best results. These results suggest that PO effectively alleviated psoriasis-like skin lesions and down-regulated inflammatory responses, which indicates that PO could potentially be used for further studies on inflammation-related skin diseases such as psoriasis and for the treatment of psoriasis such as psoriasis natural plant essential oil resources.

Presence of Cholesterol in Non-Animal Organisms: Identification and Quantification of Cholesterol in Crude Seed Oil from Perilla frutescens and Dehydrated Pyropia tenera.

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida, and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3-0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

The Contents of Polyphenols in Perilla frutescens (L.) Britton var. frutescens (Egoma) Leaves are Determined by Vegetative Stage, Spatial Leaf Position, and Timing of Harvesting during the Day.

The leaf of Perilla frutescens (L.) Britton var. frutescens (egoma) is a rich source of polyphenolic compounds, including rosmarinic acid. However, there is still a lack of detailed information concerning the content of phenolic compounds in these leaves. Since some flavonoids were found as a conjugated form, leaves were used untreated or hydrolyzed using ß-glucuronidase for analysis. Enzymatic hydrolysis method successfully identified some polyphenols, which have not been reported before. Scutellarin, a flavone glucuronide with a molecular mass similar to that of luteolin 7-O-glucuronide, was present in egoma leaves. Scutellarin was the second most abundant polyphenolic compound, after rosmarinic acid. Egoma leaves at the top of the plant contained a higher amount of rosmarinic acid and scutellarin compared to that in the leaves below. The difference in plant growth stage also influenced the rosmarinic acid and scutellarin contents, while the time of harvesting during the day did rosmarinic acid contents only. This is the first time that scutellarin, a traditional Chinese medicine, widely used for the treatment of cerebrovascular disease, was quantitatively determined in egoma leaves. The present study may help adding value to egoma leaves, developing dietary supplements, functional foods, and cosmetics.

Responses of glutathione and phytochelatins biosysthesis in a cadmium accumulator of Perilla frutescens (L.) Britt. under cadmium contaminated conditions.

Screening new accumulators of heavy metal and identifying their tolerance, enrichment capacity of heavy metals are currently hot issues in phytoremediation research. A series of hydroponic experiments were conducted to analyze the effects of glutathione and phytochelatins in roots, stems, and leaves of Perilla frutescens under cadmium stress. The results showed that the non-protein thiols in roots and stems mainly existed in the form of GSH, PC2, PC3, and PC4 under Cd stress condition, while in leaves they existed in the form of GSH, PC2, and PC3. Furthermore, the contents of GSH and PCs positively correlated with Cd, but negatively correlated with root vigor and chlorophyll content under Cd stress conditions. After 21 days of treatments, the contents of Cd in different parts of the plant were 1465.2-3092.9 mg· kg-1 in the roots, 199.6-478.4 mg·kg-1 in the stems and 61.3-96.9 mg· kg-1 in the leaves at 2, 5, 10 mg·L-1 Cd levels respectively, and the amount of Cd uptakes were up to 3547.7-5701.7 µg·plant-1. Therefore, P. frutescens performed high capacity in Cd accumulation, and PCs played a key role in Cd tolerance. The application prospect of the plant in phytoremediation Cd polluted soil was also discussed.

Identification and quantification of essential oil content and composition, total polyphenols and antioxidant capacity of Perilla frutescens (L.) Britt.

The objective of this study was to investigate the volatile compounds of the leaves of ten perilla accessions as well as to determine total polyphenols, antioxidant capacity. Essential oil (EOs) content ranged from 0.33 to 1.75 mL/100 g d.w. in PS3 and J1 respectively. In this study sixty-five compounds were identified by GC-MS and characterized with the predominance of perillaldehyde, perilla ketone, ß-dehydro-elsholtzia ketone, limonene, shisofuran, farnesene (Z, E, α), ß-caryophyllene, trans-shisool. The biogenesis and composition of EOs are probably attributed to several factors. JTD3 had a significantly higher polyphenol content as well as showed the highest antioxidant capacity, whereas a strong positive linear correlation was observed between them. PS1 and NP 606 produced the maximum biomass correspondingly, while a large glandular trichome density was recorded for J1. The results support that perilla is rich in natural compounds that could be developed as nutraceuticals and/or phytomedicine.

Characterization of metabolite profiles from the leaves of green perilla (Perilla frutescens) by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry and screening for their antioxidant properties.

The objective of this research was to access the determination of metabolite profiles and antioxidant properties in the leaves of green perilla (Perilla frutescens), where these are considered functional and nutraceutical substances in Korea. A total of 25 compositions were confirmed as six phenolic acids, two triterpenoids, eight flavonoids, seven fatty acids, and two glucosides using an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) technique from the methanol extract of this species. The individual and total compositions exhibited significant differences, especially rosmarinic acid (10), and linolenic acids (22 and 23) were detected as the predominant metabolites. Interestingly, rosmarinic acid (10) was observed to have considerable differences with various concentrations in three samples (Doryong, 6.38 µg/g; Sinseong, 317.60 µg/g; Bongmyeong, 903.53 µg/g) by UPLC analysis at 330 nm. The scavenging properties against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals also showed potent effects with remarkable differences at a concentration of 100 µg/mL, and their abilities were as follows: Sinseong (DPPH, 86%; ABTS, 90%) > Bongmyeong (71% and 84%, respectively) > Doryong (63% and 73%, respectively). Our results suggest that the antioxidant activities of green perilla leaves are correlated with metabolite contents, especially the five major compositions 10 and 22-25. Moreover, this study may be useful in evaluating the relationship between metabolite composition and antioxidant activity.

[Advances in research of volatile oil and its different chemotypes in leaves of Perilla frutescens].

The volatile oil is the main component in the leaves of Perilla frutescens. According to the main types of monoterpenoids or aromatic compounds, it can be divided into different chemotypes and the main chemotypes of Chinese producing Perilla are PA type (mainly containing Perilla aldehyde and limonene), PK type (mainly containing perillaketone) and PP type (subdivided as PP-a type, with apiole as its main component; PP-m type, with myristicin as its main component; PP-e type, with elemicin as main component; PP-as type, with asarone as main component). Based on the biosynthetic pathways analysis, we also found that the formation of the particular chemotype is usually controlled by a single gene or a few genes, and different types have different pharmacological effects. In this paper, the classification under the species P. frutescens, main chemotypes of the volatile oil, and their biogenesis and regulation, pharmacological effect and influence factors are summarized and reviewed.

Production and applications of rosmarinic acid and structurally related compounds.

Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids L-phenylalanine and L-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer's disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy.

Anti-inflammatory effects of Perilla frutescens leaf extract on lipopolysaccharide-stimulated RAW264.7 cells.

Perilla leaves are widely used in Chinese herbal medicine and in Japanese herbal agents used to treat respiratory diseases. This study aimed to investigate the anti­inflammatory effects and the underlying mechanisms of Perilla frutescens leaf extract (PLE). Murine macrophage RAW264.7 cells were used as a model. Cell viability and morphological changes were studied by the MTT assay and microscopy. mRNA expression of pro­inflammatory mediators was assessed by both semi­quantitative reverse transcription­polymerase chain reaction (RT­PCR) and quantitative (q) RT­PCR. Nitric oxide (NO) and prostaglandin E2 (PGE2) production were analyzed by the Griess test and sandwich enzyme­linked immunosorbent assay (ELISA), respectively. The activation of kinase cascades was studied by immunoblotting. Our findings showed that PLE slightly affects cell viability, but alleviates LPS­induced activation of RAW264.7 cells. Furthermore, PLE significantly reduced the LPS­induced mRNA expression of the interleukin (IL)­6, IL­8, tumor necrosis factor­α (TNF­α), cyclooxygenase­2 (COX­2) and inducible nitric oxide synthase (iNOS), genes in a dose­dependent manner. In addition, PLE reduced NO production and PGE2 secretion induced by LPS. PLE also inhibited activation of mitogen­activated protein kinases (MAPKs), increased the cytosolic IκBα level, and reduced the level of nuclear factor (NF)­κB. Taken together, these findings indicate that PLE significantly decreases the mRNA expression and protein production of pro­inflammatory mediators, via the inhibition of extracellular­signal­regulated kinase (ERK)1/2, c­Jun N­terminal kinase (JNK), p38, as well as NF­κB signaling in RAW264.7 cells stimulated with LPS.

Antidepressant-like effects of Perilla frutescens seed oil during a forced swimming test.

Unipolar depressive disorder may become one of the major leading causes of disease burden by 2030 according to the World Health Organization (WHO). Thus, the discovery of antidepressive foods is attractive and could have considerable impacts worldwide. We investigated the antidepressant-like effects of Perilla frutescens seed oil on adult male rats subjected to a forced swimming test (FST). Forty Sprague-Dawley rats were housed and fed various diets, including soybean oil-rich, eicosapentaenoic acid (EPA)-rich, and P. frutescens seed oil-rich diets for 6 weeks. After the dietary intervention, animals were tested using an FST and were sacrificed after the test. We analyzed the fatty acid profiles of red blood cells (RBCs) and the brain prefrontal cortex (PFC). Levels of brain-derived neurotrophic factor (BDNF), serotonin, and dopamine in the PFC were also determined. After the FST, the imipramine, EPA-rich, and P. frutescens seed oil-rich groups showed significant shorter immobility time and longer struggling time than the control group (p < 0.05). Levels of BDNF in the P. frutescens seed oil-rich group and levels of serotonin in the EPA-rich group were significantly (p < 0.05) higher than those of the control group. Moreover, the BDNF concentration in the PFC was significantly positively correlated with the struggling time. However, there were no significant differences in dopamine levels between the intervention groups and the control group. In conclusion, a P. frutescens seed oil-rich diet exhibited antidepressant-like properties through modulation of fatty acid profiles and BDNF expression in the brain during an FST.

Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.

Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.